Lyme disease is the most common arthropod-borne disease in the United States, with over 38,000 confirmed and probable cases reported in 2009. The causative agent of Lyme disease is the spirochete Borrelia burgdorferi, which is transmitted by the tick of the Ixodes scapularis tick. The number of Lyme disease cases has tripled over the past fifteen years. The disease is sometimes difficult to diagnose in its early stages, leading to delayed treatment. Patients who are not treated in a timely fashion may suffer from severe, long lasting symptoms, and fail to respond to antibiotic therapy. Control measures designed to prevent contact with ticks are not sufficient to prevent the disease. We propose to develop a vaccine to control Lyme disease in human populations. After being injected into the skin of a mammalian host, Borrelia bacteria multiply locally before disseminating to other organs. We have now for the first time been able to isolate sufficient amounts of bacteria from the skin of infected mice in order to identify proteins expressed at high levels in the skin. We believe that immunization against these proteins, which are expressed in the early acute phase of Lyme disease, will be highly effective in preventing bacterial proliferation and dissemination. Fifteen membrane proteins identified in the skin of infected mice will be produced in recombinant form using standard bacterial expression systems. These proteins are conserved among strains of Borrelia, which should ensure that our vaccine is effective against all strains of circulating Borrelia. Each antigen will then be administered to C57Bl/6 mice in the presence of an adjuvant. We will demonstrate that this immunization protocol results in the appearance of anti-Borrelia antibodies. Immunized animals will then be challenged with 104 live Borrelia spirochetes. Twenty-one days after challenge we will collect tissue samples to determine whether they contain live Borrelia. We will also quantitatively detect the presence of Borrelia DNA in these samples. A protein antigen will be considered protective (1) if spirochetes cannot be cultured from the tissues of immunized mice after challenge or (2) if the bacterial load, determined from the amount of DNA detected, is reduced by at least 50%. At the conclusion of these experiments we will select up to four antigens for further study. Phase II experiments will determine the optimal formulation and method of delivery. We will also examine whether antigens can be combined to provide full protection against Borrelia.